Human B-cell tolerance and autoimmunity

Even healthy subjects possess a large number of B-cells with the potential to produce autoantibodies that may cause autoimmune diseases such Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and Diabetes among others. Thus, there is a need for the proper regulation of these potentially pathogenic cells. This process is known as B-cell tolerance. Our laboratory has developed a unique system for the study of this essential process in humans. Using this system, which takes advantage of the properties of B-cells expressing antibodies encoded by the VH4-34 gene, we have been able to show that in healthy subjects autoreactive B-cells are physiologically censored in the germinal centers, the anatomical compartment within the secondary lymphoid tissue responsible for affinity maturation, isotype switch and selection of memory B-cells and long-lived plasma cells. In contrast, in SLE patients the germinal centers are defective in their ability to censor the maturation and selection of autoreactive B-cells. This defect results in the accumulation of these cells in the long-lived post-germinal center compartments (memory and plasma cells) where they have a central contribution to the pathogenesis of the disease. The identification of this defect has been enabled by our ability to perform tonsil biopsies in autoimmune patients, a unique resource not readily available in most medical centers. These studies represent the first identification of a defective check point in the maintenance of B-cell tolerance in human SLE. Interestingly, this defect is not shared by patients with RA and therefore it may hold important clues for our understanding of Lupus and other autoimmune diseases. Currently, we are pursuing a multidisciplinary approach to understand the cellular and molecular bases that underlie the regulation of B-cell tolerance. Our approach includes a systematic study of gene expression profiles in finely discriminated B-cell subsets as well as signaling studies (see below).

Studies of human B-cell development and function

Our laboratory routinely characterizes a large number of discreet subsets of human B-cells on the basis of their surface phenotype using multi-parameter flow cytometry and immuno-histology. Using these approaches we have also described two new subsets of human B-cells that represent a novel fraction of memory cells and circulating marginal zone B-cells. More importantly, we have recently identified a highly proliferative population of tonsil B-cells that represent the founder cells of the germinal center reactions. Interestingly, this founder population represents the developmental stage at which autoreactive B-cells are censored in healthy subjects but not in SLE patients. Therefore, we are using microarray analysis of gene transcription in these populations to identify the genetic programs responsible for the development of these critical B-cell subsets and their abnormal regulation in autoimmune diseases.

B-cell depletion for the therapy of autoimmune diseases

Our group is one of the pioneers in the use of in vivo B-cell depletion (using the anti-CD20 monoclonal antibody Rituximab) for the therapy of autoimmune diseases. We have recently completed the first phase I/II clinical trial of Rituximab in SLE in which we have demonstrated that clinical improvement is associated with B-cell depletion and may be unrelated to changes in autoantibody levels thereby suggesting that B-cells play also important immunoregulatory functions that are independent of antibody production. Our studies also indicate B-cell depletion may offer the immune system a second chance to re-establish B-cell tolerance and therefore hold promise for the long-term treatment and prevention of autoimmune diseases. These studies are performed in collaboration with Dr. Looney (Principal Investigator for the clinical trials) and Dr. Anolik who is also a central collaborator in the studies described previously. In the near future we are planning to use B-cell depletion for the treatment/prevention of other autoimmune diseases including Diabetes (in collaboration with Drs. Looney and Jospe) and Rheumatoid Arthritis.

Innovative approaches to the study and treatment of autoimmune diseases

The University of Rochester is home to one of only seven national Autoimmunity Centers of Excellence (ACE) funded in the nation by the NIH. Dr. Sanz is the Center Director. The Rochester ACE also includes Drs. Looney (Medicine-Rheumatology/SLE), Segal, Goodman and Schwid (Neurology/Multiple Sclerosis), Fowel (Vaccine Biology, Diabetes), Jospe (Pediatrics/Diabetes) and Mosmann (Vaccine Biology and Core Director for the ACE). In addition to these investigators and diseases, the ACE center works with the other ACEs to develop several protocols every year to support innovative approaches to the study and therapy of a wide variety of autoimmune diseases. The ACE center thus provides a unique opportunity for people investigators and students interested in participating in a multidisciplinary approach to human autoimmunity encompassing both clinical and basic research

Milner EC, Anolik J, Cappione A, Sanz I. Human innate B cells: a link between host defense and autoimmunity? Springer Semin Immunopathol. 2005 Jan 5; [Epub ahead of print]

Looney RJ, Anolik J, Sanz I. Treatment of SLE with anti-CD20 monoclonal antibody. Curr Dir Autoimmun. 8:193-205, 2005.

Anolik J, Barnard J, Cappione A, Pugh-Bernard A, Felgar R, Looney RJ, Sanz I. Rituximab improves peripheral B cell abnormalities in human SLE. Arthritis & Rheumatism, 2004. Arthritis Rheum. Nov;50(11):3580-90, 2004.

Looney, R. J., A. J., D. Campbell, R. E. Felgar, F. Young, L. J. Arend, J. A. Sloand, J. Rosenblatt, and I. Sanz. 2004. B cell depletion as a novel treatment for systemic lupus erythematosus: A phase I/II dose-escalation trial of rituximab. Arthritis & Rheumatism 50:2580, 2004.

Anolik J, Sanz, I. B-cells in human and murine SLE. Curr Opin Rheumatol. 16: 505-512, 2004

Looney RJ, Anolik J, Sanz I. B lymphocytes in Systemic Lupus Erythematosus: lessons from therapy targeting B cells. Lupus 13:1-10, 2004.

Looney RJ, Anolik J, Sanz I. B cells as therapeutic targets for rheumatic diseases. Curr Opin Rheumatol. 16:180-5, 2004.

Cappione AJ, Pugh-Bernard AE, Anolik JH, Sanz I. Lupus IgG V(H)4.34 antibodies bind to a 220-kDa glycoform of CD45/B220 on the surface of human B lymphocytes. J Immunol. 172:4298-307, 2004.

Zhang MY, Shu Y, Phogat S, Xiao X, Cham F, Bouma P, Choudhary A, Feng YR, Sanz I, Rybak S, Broder CC, Quinnan GV, Evans T, Dimitrov DS. Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library. J Immunol Methods. 283:17-25, 2003.

Anolik J, Sanz I, Looney RJ. B cell depletion therapy in systemic lupus erythematosus. Curr Rheumatol Rep. 5:350-6, 2003.

Anolik J, Looney RJ, Bottaro A, Sanz I, Young F. Down-regulation of CD20 on B cells upon CD40 activation. Eur J Immunol. 33:2398-409, 2003.

Anolik JH, Campbell D, Felgar RE, Young F, Sanz I, Rosenblatt J, Looney RJ. The relationship of FcgammaRIIIa genotype to degree of B cell depletion by rituximab in the treatment of systemic lupus erythematosus. Arthritis Rheum. 48:455-459, 2003.

Pugh-Bernard AE, Silverman GJ, Cappione AJ, Villano ME, Ryan DH, Insel RA, Sanz I. Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance. J Clin Invest. 108:1061-70, 2001.

del Rincon I, Zeidel M, Rey E, Harley JB, James JA, Fischbach M, Sanz I. Delineation of the human systemic lupus erythematosus anti-smith antibody response using phage-display combinatorial libraries. J Immunol. 165:7011-6, 2000.

Bili A, Moss AJ, Francis CW, Zareba W, Watelet LF, Sanz I. Anticardiolipin antibodies and recurrent coronary events: a prospective study of 1150 patients. Thrombogenic Factors, and Recurrent Coronary Events Investigators. Circulation 102:1258-63, 2000.

Rey E, Zeidel M, Rhine C, Tami J, Krolick K, Fischbach M, Sanz I. Characterization of human anti-acetylcholine receptor monoclonal autoantibodies from the peripheral blood of a myasthenia gravis patient using combinatorial libraries. Clin Immunol. 96:269-79, 2000.

Li P, Sanz I, O'Keefe RJ, Schwarz EM. NF-kappa B regulates VCAM-1 expression on fibroblast-like synoviocytes. J Immunol. 164:5990-7, 2000.

Goater J, Muller R, Kollias G, Firestein GS, Sanz I, O'Keefe RJ, Schwarz EM. Empirical advantages of adeno associated viral vectors in vivo gene therapy for arthritis. J Rheumatol. 27:983-9, 2000.

Pryhuber GS, O'Brien DP, Baggs R, Phipps R, Huyck H, Sanz I, Nahm MH. Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury. Am J Physiol Lung Cell Mol Physiol. 278:L1082-90, 2000.

Deng SX, Hanson E, Sanz I. In vivo cell penetration and intracellular transport of anti-Sm and anti-La autoantibodies. Int Immunol. 12:415-23, 2000.

O'Brien DP, Briles DE, Szalai AJ, Tu AH, Sanz I, Nahm MH. Tumor necrosis factor alpha receptor I is important for survival from Streptococcus pneumoniae infections. Infect Immun 67:595-601, 1999.